The major goal of the proposed project is to study how human epithelial cells become immortal. Cellular immortalization is a key event in the development of malignancy, and epithelial cells give rise to the majority of human cancers. The hypothesis to be tested is that both the abrogation of the retinoblastoma pathway and activation of telomerase is necessary for epithelial cells to overcome senescence. By using unique epithelial cell model systems developed in the laboratory, the PI proposes to identify factors involved in the induction of the Rb pathway during senescence and determine how telomerase is activated as cells become immortal. The role of terminal differentiation in activation of the Rb pathway and senescence will be examined by several approaches, including regulated expression of p16INK4a, a cdk4/cyclin D inhibitor, in epithelial cells that have specifically lost p16INK4a expression upon immortalization with hTERT, the catalytic component of telomerase. Genes involved in upregulation of p16INK4a will also be identified by using cDNA retroviral libraries to bypass the Rb mediated block. Because expression of p16INK4a is commonly lost in human cancers, the PI observes that this work is likely to lead to a significant increase in our understanding of the early stages of carcinogenesis. Previous studies demonstrated that human pappiloma virus E6 activates telomerase. To determine the mechanism of this activation, HPV E6 mutants that differentially activate telomerase will be used in cDNA expression array analysis and yeast two-hybrid screens to identify factors involved in telomerase activation by HPV E6. Understanding how E6 activates telomerase will be useful in elucidating how telomerase is activated in general. The PI observes that these studies have the potential of leading to new and innovative strategies for the prevention, diagnosis and treatment of human cancer. The overall goal of this proposal is to study how human epithelial cells become immortal, which is a key event in the development of malignancy. The hypothesis central to this proposal is that both abrogation of the retinoblastoma pathway and activation of telomerase are necessary for epithelial cells to overcome senescence. By using unique epithelial cell model systems developed in his laboratory the PI will pursue the following specific aims: (1) to determine the role of differentiation in Rb-mediated senescence of human epithelial cells; (2) to identify and characterize upstream genes involved in induction of Rb-mediated senescence in human epithelial cells; and (3) to determine how HPV 16 E6 activates telomerase in epithelial cells. These studies should have the potential of leading to new and innovative strategies for the prevention, diagnosis and treatment of human cancer.